PROJECT SUMMARY The development, functional activity, and plasticity of neuronal circuits rely critically on the spatially controlled expression and regulation of synaptic proteins and their messenger RNAs (mRNAs). Genome-wide association studies have revealed extensive polygenic variation in synaptic proteins in association with diseases including autism, schizophrenia, and Alzheimer's. A molecular understanding of how these complex genetic variations impact neuronal synapse development, plasticity, and homeostasis is crucial for the development of new therapies to treat these diseases. Fluorescence imaging offers the potential to characterize neuronal synapse protein and mRNA levels and localizations in situ; however, current imaging approaches can simultaneously interrogate no more than four of the several dozen molecules of interest in any given neuronal sample. To overcome this obstacle, we propose to develop a transformative fluorescence imaging assay that enables simultaneous, highly multiplexed, high-throughput molecular characterization of protein and mRNA expression levels and localizations in intact neurons. To this end, we will develop an innovative labeling strategy that exploits transiently binding fluorescent nucleic acids to enable multiple rounds of imaging of intact specimens, using both standard and super-resolution microscopy. In conjunction, we will develop ultra-bright fluorescent probes based on hybridization chain reaction and structured nucleic acids for visualization of single mRNA molecules. We will apply both standard confocal and super-resolution imaging to characterize spatial distributions and molecular interactions of synaptic proteins and regulatory mRNA-binding proteins, including Fragile-X Mental Retardation Protein (FMRP) in both mouse and human induced pluripotent stem cell models. Using this approach, we will characterize the impact of gene deletions associated with autism on the levels and localizations of more than 10 synaptic and cytoskeletal proteins, as well as examine the interactions of FMRP with dozens of mRNAs in intact dendritic arbors, spines, and synapses. We intend our technique to become broadly useful as a platform technology for the study of the molecular impacts of genetic variations in psychiatric diseases, including autism and schizophrenia. Consequently, we will develop our imaging platform in close collaboration with the Stanley Center at the Broad Institute of MIT and Harvard. The high-throughput nature of our imaging approach ensures that it will be useful for development of novel methods of treating psychiatric diseases using small-molecule and gene-editing approaches.